title: Purification of human midcycle cervical mucin and characterization of its oligosaccharides with respect to size, composition, and microheterogeneity.
abstract: Human cervical mucin was solubilized from the gel phase of pooled midcycle cervical mucus using 6 M guanidine hydrochloride and 10 mM dithiothreitol and was then alkylated with iodoacetamide. Mucin was then purified by gel filtration on Bio-Gel A-50m resin in buffer containing 0.1% sodium dodecyl sulfate. The purified mucin gave a single band upon electrophoresis in either 5% acrylamide or 1% agarose gels. Protein comprised 21% of the glycoprotein by weight and amino acid analysis revealed a high content of Ser and Thr. Saccharide analysis yielded approximate molar ratios of Fuc:Gal:GlcNAc:GalNAc:NeuAc = 1:2:1:1:0.5. Inorganic sulfate, 1% by weight, was detected, but mannose was absent. Reductive alkali treatment of mucin resulted in release of oligosaccharides with concomitant conversion of 77% of GalNAc to its reduced derivative N-acetylgalactosaminitol (GalNAcol) thus demonstrating O-glycosidic linkage of GalNAc to protein. Reduced oligosaccharides were purified by ion exchange chromatography on DEAE-cellulose, paper chromatography, and high resolution gel filtration on Bio-Gel P-2 resin. A total of 16 reduced oligosaccharides were identified by thin layer chromatography. These included neutral, sialylated, and sulfated oligosaccharides and they varied in size from a disaccharide to a nonasaccharide. The major neutral oligosaccharide isolated (21% of recovered GalNAcol) was a tetrasaccharide, Gal:GlcNAc:GalNAcol = 2:1:1, and the major acidic oligosaccharide isolated (11% of recovered GalNAcol) was a trisaccharide, Gal:GalNAcol:NeuAc = 1:1:1.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002584:Cervix Uteri / Q000032:analysis*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D005779:Immunodiffusion; D037102:Lectins; D008598:Menstruation; D009077:Mucins / Q000302:isolation & purification*; D009844:Oligosaccharides / Q000032:analysis*; D012995:Solubility
chemical_list: D000596:Amino Acids; D002241:Carbohydrates; D037102:Lectins; D009077:Mucins; D009844:Oligosaccharides